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knock-in mice have been previously described [12]. Littermate pups from heterozygote crosses were used for primary culture establishment, by previously described methods that yield cerebellar granule neuron-enriched cultures [26]. Postnatal day 4 (P4) cerebella were dissected in Hank's Balanced Salt Solution (HBSS, Sigma), supplemented with 35 mM glucose. Tail biopsies were also collected for genomic DNA isolation and genotypic analysis. Trypsin/EDTA (10 mg/ml, Sigma) and DNase I (100 μg/ml, Sigma), suspended in HBSS, helped dissociate cerebella for primary culture plating onto 0.01% poly-ornithine (Sigma) coated 100 mm dishes. Primary cultures from individual cerebella were cultured overnight at 37°C, 5% CO2, in granule neuron growth media (Dulbecco's Modified Eagle Medium [DMEM, Gibco BRL #11995-065], 10% fetal bovine serum [Sigma #F-2442], supplemented with 24 mM KCl). Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [27], as previously described [28]. Following infection, cultures were shifted to the tsA58/U19 permissive growth temperature of 33°C and selection was in the same growth media as above, with 200 μg/ml G418. Conditionally immortalized colonies were isolated 3–9 weeks post-infection and expanded for frozen stocks and further sub-clone isolation. Multiple clonal lines were obtained for each genotype and all phenotypes were confirmed in at least 2 independent CbCln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [29] with the following cocktail: 10 ng/ml α-FGF, 250 μM IBMX, 200 nM TPA, 50 μM forskolin, 5 μM dopamine (Sigma)."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T5663","span":{"begin":0,"end":4},"obj":"PR_EXT:000005591"},{"id":"T5664","span":{"begin":20,"end":24},"obj":"NCBITaxon:10088"},{"id":"T5665","span":{"begin":73,"end":77},"obj":"NCBITaxon_UBERON_EXT:pup"},{"id":"T5666","span":{"begin":192,"end":202},"obj":"UBERON:0002037"},{"id":"T5667","span":{"begin":192,"end":217},"obj":"CL:0001031"},{"id":"T5668","span":{"begin":246,"end":251},"obj":"GO:0007567"},{"id":"T5669","span":{"begin":263,"end":272},"obj":"UBERON:0002037"},{"id":"T5670","span":{"begin":307,"end":320},"obj":"CHEBI_EXT:saline_solution"},{"id":"T5671","span":{"begin":360,"end":367},"obj":"CHEBI:17234"},{"id":"T5672","span":{"begin":369,"end":373},"obj":"UBERON:0002415"},{"id":"T5673","span":{"begin":407,"end":418},"obj":"SO_EXT:genomic_DNA"},{"id":"T5674","span":{"begin":415,"end":418},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T5675","span":{"begin":433,"end":442},"obj":"SO_EXT:genotype_or_entity_with_genotype"},{"id":"T5676","span":{"begin":453,"end":460},"obj":"PR_EXT:000027795"},{"id":"T5677","span":{"begin":461,"end":465},"obj":"CHEBI_EXT:EDTA"},{"id":"T5678","span":{"begin":488,"end":495},"obj":"GO_PR_EXT:deoxyribonuclease_I"},{"id":"T5679","span":{"begin":553,"end":562},"obj":"UBERON:0002037"},{"id":"T5680","span":{"begin":607,"end":616},"obj":"CHEBI_EXT:ornithine"},{"id":"T5681","span":{"begin":680,"end":689},"obj":"UBERON:0002037"},{"id":"T5682","span":{"begin":726,"end":729},"obj":"CHEBI:16526"},{"id":"T5683","span":{"begin":734,"end":748},"obj":"CL:0000120"},{"id":"T5684","span":{"begin":749,"end":755},"obj":"GO_EXT:biological_growth_entity_or_process"},{"id":"T5685","span":{"begin":836,"end":842},"obj":"NCBITaxon:27592"},{"id":"T5686","span":{"begin":843,"end":848},"obj":"UBERON:0001977"},{"id":"T5687","span":{"begin":890,"end":893},"obj":"CHEBI:32588"},{"id":"T5688","span":{"begin":953,"end":963},"obj":"NCBITaxon:11632"},{"id":"T5689","span":{"begin":964,"end":975},"obj":"GO:0009293"},{"id":"T5690","span":{"begin":1020,"end":1027},"obj":"CHEBI_EXT:59132"},{"id":"T5691","span":{"begin":1044,"end":1052},"obj":"CHEBI:7507"},{"id":"T5692","span":{"begin":1064,"end":1072},"obj":"SO_EXT:0005853"},{"id":"T5693","span":{"begin":1180,"end":1186},"obj":"GO_EXT:biological_growth_entity_or_process"},{"id":"T5694","span":{"begin":1237,"end":1243},"obj":"GO_EXT:biological_growth_entity_or_process"},{"id":"T5695","span":{"begin":1275,"end":1279},"obj":"CHEBI:42768"},{"id":"T5696","span":{"begin":1429,"end":1435},"obj":"SO_EXT:sequence_cloned_entity"},{"id":"T5697","span":{"begin":1465,"end":1473},"obj":"SO_EXT:genotype_or_entity_with_genotype"},{"id":"T5698","span":{"begin":1534,"end":1536},"obj":"UBERON:0002037"},{"id":"T5699","span":{"begin":1536,"end":1540},"obj":"PR_EXT:000005591"},{"id":"T5700","span":{"begin":1541,"end":1545},"obj":"CL_GO_EXT:cell"},{"id":"T5701","span":{"begin":1553,"end":1555},"obj":"UBERON:0002037"},{"id":"T5702","span":{"begin":1560,"end":1564},"obj":"CL_GO_EXT:cell"},{"id":"T5703","span":{"begin":1601,"end":1610},"obj":"CHEBI_EXT:ornithine"},{"id":"T5704","span":{"begin":1662,"end":1665},"obj":"CHEBI:16526"},{"id":"T5705","span":{"begin":1769,"end":1777},"obj":"CL:0000540"},{"id":"T5706","span":{"begin":1778,"end":1793},"obj":"GO_RO_EXT:developmental_differentiation_process"},{"id":"T5707","span":{"begin":1846,"end":1854},"obj":"CHEBI:60004"},{"id":"T5708","span":{"begin":1865,"end":1870},"obj":"PR_EXT:000007479"},{"id":"T5709","span":{"begin":1879,"end":1883},"obj":"CHEBI:48518"},{"id":"T5710","span":{"begin":1892,"end":1895},"obj":"CHEBI:37537"},{"id":"T5711","span":{"begin":1903,"end":1912},"obj":"CHEBI:42471"},{"id":"T5712","span":{"begin":1919,"end":1927},"obj":"CHEBI:18243"}],"text":"Cln3Δex7/8 knock-in mice have been previously described [12]. Littermate pups from heterozygote crosses were used for primary culture establishment, by previously described methods that yield cerebellar granule neuron-enriched cultures [26]. Postnatal day 4 (P4) cerebella were dissected in Hank's Balanced Salt Solution (HBSS, Sigma), supplemented with 35 mM glucose. Tail biopsies were also collected for genomic DNA isolation and genotypic analysis. Trypsin/EDTA (10 mg/ml, Sigma) and DNase I (100 μg/ml, Sigma), suspended in HBSS, helped dissociate cerebella for primary culture plating onto 0.01% poly-ornithine (Sigma) coated 100 mm dishes. Primary cultures from individual cerebella were cultured overnight at 37°C, 5% CO2, in granule neuron growth media (Dulbecco's Modified Eagle Medium [DMEM, Gibco BRL #11995-065], 10% fetal bovine serum [Sigma #F-2442], supplemented with 24 mM KCl). Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [27], as previously described [28]. Following infection, cultures were shifted to the tsA58/U19 permissive growth temperature of 33°C and selection was in the same growth media as above, with 200 μg/ml G418. Conditionally immortalized colonies were isolated 3–9 weeks post-infection and expanded for frozen stocks and further sub-clone isolation. Multiple clonal lines were obtained for each genotype and all phenotypes were confirmed in at least 2 independent CbCln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [29] with the following cocktail: 10 ng/ml α-FGF, 250 μM IBMX, 200 nM TPA, 50 μM forskolin, 5 μM dopamine (Sigma)."}

{"project":"2_test","denotations":[{"id":"15588329-12374761-12950162","span":{"begin":57,"end":59},"obj":"12374761"},{"id":"15588329-8248201-12950163","span":{"begin":237,"end":239},"obj":"8248201"},{"id":"15588329-3023876-12950164","span":{"begin":1074,"end":1076},"obj":"3023876"},{"id":"15588329-9671979-12950165","span":{"begin":1104,"end":1106},"obj":"9671979"},{"id":"15588329-11092756-12950166","span":{"begin":1823,"end":1825},"obj":"11092756"}],"text":"Cln3Δex7/8 knock-in mice have been previously described [12]. Littermate pups from heterozygote crosses were used for primary culture establishment, by previously described methods that yield cerebellar granule neuron-enriched cultures [26]. Postnatal day 4 (P4) cerebella were dissected in Hank's Balanced Salt Solution (HBSS, Sigma), supplemented with 35 mM glucose. Tail biopsies were also collected for genomic DNA isolation and genotypic analysis. Trypsin/EDTA (10 mg/ml, Sigma) and DNase I (100 μg/ml, Sigma), suspended in HBSS, helped dissociate cerebella for primary culture plating onto 0.01% poly-ornithine (Sigma) coated 100 mm dishes. Primary cultures from individual cerebella were cultured overnight at 37°C, 5% CO2, in granule neuron growth media (Dulbecco's Modified Eagle Medium [DMEM, Gibco BRL #11995-065], 10% fetal bovine serum [Sigma #F-2442], supplemented with 24 mM KCl). Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [27], as previously described [28]. Following infection, cultures were shifted to the tsA58/U19 permissive growth temperature of 33°C and selection was in the same growth media as above, with 200 μg/ml G418. Conditionally immortalized colonies were isolated 3–9 weeks post-infection and expanded for frozen stocks and further sub-clone isolation. Multiple clonal lines were obtained for each genotype and all phenotypes were confirmed in at least 2 independent CbCln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [29] with the following cocktail: 10 ng/ml α-FGF, 250 μM IBMX, 200 nM TPA, 50 μM forskolin, 5 μM dopamine (Sigma)."}